May/June 2001 ⅐ Vol. 3 ⅐ No. 3 article

Modifier for mitochondrial DNA disease: Linkage and linkage disequilibrium mapping of a nuclear modifier for maternally inherited deafness Yelena Bykhovskaya, MS1, Huiying Yang, MD, PhD1, Kent Taylor, PhD1, Tieu Hang, BA1, Richard Y.M. Tun, PhD1, Xavier Estivill, MD, PhD2, Rosaria A.M.S. Casano, PhD3, Kari Majamaa, MD4, Mordechai Shohat, MD 5, and Nathan Fischel-Ghodsian, MD1 Purpose: To examine the role of the nuclear in affecting the phenotypic expression of the simplest model of a mitochondrial DNA disease, maternally transmitted deafness. Methods: Linkage analysis in families with maternally inherited deafness associated with the homoplasmic A1555G . Results: Significant linkage and linkage disequilibrium on 8 was identified. Conclusions: This finding represents the first identification of a modifier locus for a mitochondrial DNA disease and supports the concept of mitochondrial DNA diseases having complex genetic inheritance. The eventual identification of this modifier gene will provide insights into the pathophysiological pathways determining the clinical expression of mitochondrial DNA diseases, an important step toward diagnostic and therapeutic interventions. Genetics in Medicine, 2001:3(3):177–180. Key Words: mitochondrial mutation, deafness, nuclear modifier gene, linkage analysis, linkage disequilibrium

Mitochondrial DNA diseases are associated with a wide mic mitochondrial DNA . We have postulated, range of human diseases, including systemic and tissue-spe- based on biochemical and genetic analyses, that nuclear-en- cific neuromuscular disorders, diabetes mellitus, skin lesions, coded modifier are the main explanation for the pheno- aplastic anemia, and aging in general.1 Despite the clinical im- typic differences within families and that identification of these portance of these diseases, and despite the fact that the pre- modifier genes will provide the basis for diagnostic and even- sumed function of the mitochondrial chromosome have been tual therapeutic interventions.3–5 completely described for nearly two decades, the molecular Two genome-wide linkage studies in a large Arab-Israeli mechanisms leading from genotype to clinical phenotype re- family and Spanish families with the A1555G mutation led to main unknown. This has prevented useful counseling of most the conclusion that complex inheritance of nuclear modifier patients with mitochondrial DNA mutations, and has also pre- loci is responsible for clinical penetrance of the disease.6,7 Ap- vented the rational search for therapeutic interventions. To plication of nonparametric linkage analysis to the genotyping shed light on the pathophysiological pathways between geno- data of the combined Arab-Israeli, Spanish, and Italian fami- type to phenotype, we have concentrated on one of the sim- lies led to a combined allele-sharing LOD score of 3.1 at locus plest mitochondrial DNA disorders, the inherited homoplas- D8S277, a highly suggestive linkage result.7 mic A1555G mutation in the 12S rRNA gene.2 This mutation is To further investigate involvement of this region, we col- associated with nonsyndromic deafness and, despite being ho- lected an independent set of seven Spanish, Italian, and Finnish moplasmic, exhibits the same tissue specificity and great vari- families and typed original markers from the ation in clinical expression as the more common heteroplas- region in all available individuals. Addition of the new families increased the resulting multipoint LOD score from 3.1 to 4.1,

From the 1Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, thus confirming initial linkage result. Subsequent typing of Medical Genetics Birth Defects Center, Cedars-Sinai Medical Center and UCLA School of additional markers and linkage disequilibrium testing revealed Medicine, Los Angeles, California; 2Deafness Genetics Research Group, Medical and Molec- significant linkage disequilibrium with D8S277 and two adja- ular Genetics Center, Institut de Recerca Oncologica, Hospital Duran I Reynals, Barcelona, cent markers. Spain; 3Cytogenetic and Genetic Unit, Azienda Ospedaliera Careggi, Florence, Italy; 4De- partment of Neurology, University of Oulu, Oulu, Finland; and 5Department of Pediatrics and Medical Genetics, Rabin Medical Center and Basil and Gerald Felsenstein Medical SUBJECTS AND METHODS Research Center, Tel Aviv University Medical School, Petah Tikva, Israel. Pedigrees Nathan Fischel-Ghodsian, MD, Department of Pediatrics, Suite 1165WT, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. We obtained 47 from members of five multiplex fam- Received: February 1, 2001. ilies from Spain (F1–5), one multiplex family from Italy (F6), Accepted: February 23, 2001. and one nuclear family from Finland (F7) with matrilineal

Genetics IN Medicine 177 Bykhovskaya et al. nonsyndromic hearing loss (Fig. 1). Each of the families con- quencies were used for the analysis. A genetic map of the region tained at least one pair of affected siblings, and aminoglycoside was established combining sex-average map distances reported exposure was noted only in one family (F1) from Spain. Pedi- by the Marshfield Medical Research Foundation with physical gree structures of the Arab-Israeli family, and eight Spanish map data from the nonredundant (nr) and draft (htgs) Gen- and two Italian families were described previously.6,7 Informed Bank databases. consent for DNA analysis had been obtained from all members of the Spanish, Italian, and Arab-Israeli families who partici- Transmission Disequilibrium Test pated in the study. The Transmission Disequilibrium Test10 (TDT) was per- formed using both the GENEHUNTER 2.0 program5,8 and Markers TDTEX program of the SAGE package release 3.1.11,12 The Fluorescently labeled polymerase chain reaction (PCR) TDT implemented in the GENEHUNTER 2.0 tests each allele primers for markers D8S561 and D8S1469 were purchased individually and can analyze single marker, as well as two-, from Research Genetics, Inc. (Huntsville, AL). STS sequences three-, and four-marker haplotypes. Permutation test for de- corresponding to polymorphic microsatellite repeats D8S518, termining empirical significance levels was implemented. The D8S1798, D8S1742, D8S1819, D8S1140, D8S1825, D8S351 TDTEX evaluates multialleles simultaneously and can incor- were obtained from the nonredundant GenBank database, and porate sibpairs and analyze maternal and paternal transmis- PCR primers were designed using the OLIGO 6.3 program sion separately.13 (Molecular Biology Insights, Inc., Cascade, CO). These PCR primers were synthesized with fluorescent tags using the cus- RESULTS tom oligo synthesis service of Applied Biosystems (Foster City, CA). Polymerase chain reaction was done according to stan- Linkage analysis of the candidate region around D8S277 dard protocols in GeneAmp PCR system 9600 (Perkin-Elmer, We obtained DNA from seven additional families with 47 Norwalk, CT). Electrophoresis of amplified DNA was done on individuals and 21 affected sib-pairs from Spain, Italy, and a 377 automated DNA Sequencer (Applied Biosystems). Semi- Finland (see Fig. 1). First these families were genotyped for the automated DNA fragment sizing was performed using GE- seven markers in the candidate region around marker D8S277, NESCAN௣ 3.1 software, and genotyping was carried out using which were used previously to identify the suggestive linkage GENOTYPER௣ 2.5 software (Applied Biosystems). result in the region.7 These new families were combined with the previously described Arab-Israeli, Spanish, and Italian Linkage analysis families for linkage analysis. The resulting maximized allele- Multipoint nonparametric linkage analysis sharing LOD score increased from 3.1 in the previously de- To calculate nonparametric multipoint allele-sharing LOD scribed families to 4.1 (Table 1) in the combined dataset, thus scores, we used the GENEHUNTER-PLUS (ASM 1.0) pro- confirming the initial suggestive linkage result. gram.8,9 All the pedigrees included in the analysis were divided In order to more precisely localize the putative modifier into nuclear families, and the scoring function was chosen to gene in this region, additional polymorphic markers in the be “pairs.” Equal and randomly generated marker allele fre- region were typed. A map of the region was established using data from the finished sequence nonredundant GenBank da- tabase, as well as from the more preliminary htgs GenBank draft database. We genotyped an additional nine polymorphic

Table 1 Results of the nonparametric linkage analysis for the seven markers in the candidate region around D8S277 Multipoint nonparametric LOD score Distance Previously All available Marker (cM) described families families 8pter 0 D8S264 1 2.2 2.0 D8S262 5 1.8 1.5 D8S277 9 3.1 4.1 D8S503 17 1.7 1.3 D8S265 23 1.4 1.1 Fig. 1 Pedigree structure of seven families from Spain (families F1–5), Italy (F6), and Finland (F7). Hearing-impaired individuals are indicated by filled symbols. Asterisks D8S552 28 1.1 0.8 indicate individuals from whom DNA samples were obtained. Filled symbols with opened D8S1145 39 0.2 0.1 circles denote individuals who became deaf after aminoglycoside exposure.

178 Genetics IN Medicine Modifier locus for mitochondrial DNA disease microsatellite markers in the candidate region between telo- Table 2 meric marker D8S264 at 1 cM, and centromeric marker Linkage disequilibrium results for the markers in the candidate region around D8S277 D8S503 at 17 cM (D8S277 is at 9 cM) in our existing Arab- Israeli/Spanish/Italian/Finnish family set. The results are Distancea shown in Figure 2 and indicate a narrow linkage peak in the Marker (mB/cM) TDT P value Associated allele region of markers D8S277-D8S561-D8S1819 with a maximum D8S264 1.0 0.06 1 LOD score of 4.0. D8S518 2.5 0.09 4 According to the recently assembled genomic data in this D8S262 3.8 0.2 2 region, D8S277 and D8S1819 appear to be 300 kb apart. The exact location of the marker D8S561 remains not entirely clear D8S1798 5.2 0.2 10 but mostly likely is at 170 kb from D8S277. Same source of D8S1742 6.1 0.4 6 genomic data placed next adjacent marker D8S1742, previ- D8S277 9.7 0.008 8 ously considered to be at 3.6 mb away from D8S277, at only D8S561 9.9 0.01 2 300 kb distance. D8S1742 showed significantly lower multi- point LOD score of 2.1. The adjacent marker D8S1140 on the D8S1819 10.0 0.0002 6 other side of the peak with a multipoint LOD score of 1.4 still D8S1140 12.0 0.06 6 has not been located on the sequence of this region and, there- D8S1825 13.7 0.08 9 fore, is considered to be 2 cM away according to the linkage D8S351 14.1 0.2 11 maps. To assess potential variability of the LOD scores depending D8S1469 15.5 0.2 2 on marker allele frequencies, we incorporated 10 sets of ran- D8S503 16.4 0.1 3 dom marker allele frequencies, as well as CEPH families allele D8S265 23.0 0.3 4 frequencies. The values of the NPL scores and LOD scores were D8S552 28.0 0.3 8 not significantly different from the result using equal marker allele frequencies. D8S1145 39.0 0.1 6 Markers with significant linkage disequilibrium results are highlighted in bold. Using the NPL score as the measure of linkage, we observed a Ͻ Distances are given in megabases, or in centimorgans if physical distances are that nine families had a NPL score 0, likely unlinked to the not available. chromosome 8 locus, and 19 families had NPL Ͼ 0.8, poten- tially linked to the region. No families with NPL scores between 0 and 0.8 were observed. type analysis did not improve the significance level of the asso- ciations. Not all families with NPL scores over 0.8 showed ev- Linkage disequilibrium testing of the candidate region by TDT idence of association with allele 6 of D8S1819. Each of the markers typed for linkage was also tested for Exact TDT tests done by TDTEX program analyzing mul- linkage disequilibrium by the TDT using GENEHUNTER 2.0 tialleles simultaneously also independently confirmed this sta- and TDTEX programs. Using GENEHUNTER 2.0, all three tistically significant result for the markers D8S561 (permuta- markers with the most significant LOD scores also showed tion McNemar P value ϭ 0.004) and D8S1819 (P value ϭ significant linkage disequilibrium with P values as low as 0.005). The results were not significantly different when the 0.0002 for the marker D8S1819 and allele 6 (Table 2). Haplo- analyses were based on affected individual sibs or sibpairs nor when maternal and paternal transmission was analyzed sepa- rately or together.

DISCUSSION Genetic analysis of the previously suggested candidate re- gion around marker D8S277 using additional families provides significant evidence for the first human nuclear modifier locus for maternally inherited deafness. The obtained allele sharing LOD score of 4.1 for the initial markers, and 4.0 for all typed markers, has exceeded the threshold of significant linkage (LOD ϭ 3.6) proposed for genome-wide linkage screens by Lander and Kruglyak.14 Similar results have recently been ob- tained in mice, when a maternal effect was noted in the (A/J ϫ CAST/Ei) ϫ A/J backcross, with A/J being a hearing-impaired laboratory inbred strain and CAST/Ei being a normal hearing Fig. 2 Multipoint output from the GENEHUNTER program for markers in the candi- date region around D8S277. Maximized allele-sharing LOD scores are typed above the wild-derived inbred strain. The maternal effect was shown to graph. be due to a nucleotide insertion in the mitochondrial tRNA-

May/June 2001 ⅐ Vol. 3 ⅐ No. 3 179 Bykhovskaya et al.

Arg gene and was only expressed in mice homozygous for the gions will shed light on the pathophysiological pathways in- nuclear Ahl locus.15 The Ahl locus had previously been shown volved in the clinical expression of the A1555G mutation. to be a major determinant of hearing loss in the A/J strain and Acknowledgments a number of other inbred strains.16 The gene responsible for the Ahl locus effect has not yet been identified. Thus, it appears We gratefully acknowledge support by grants RO1DC01402 that, both in mouse and man, homoplasmic mitochondrial and RO1DC04092 from the National Institutes of Health/Na- DNA mutations can cause hearing impairment only in the tional Institute on Deafness and Other Communication Dis- presence of the appropriate environmental factors17 or nuclear orders, Bethesda, MD, and by The Fundacio La Marato de TV3 background. (XE). Genotyping was supported by NCRR grant MO1 Identification of the gene in the chromosome 8 region will RR00425 to the GCRC phenotyping/genotyping core at Ce- most likely require genetic fine mapping and subsequent can- dars-Sinai Medical Center. didate gene analysis. For a complex genetic disease, identifica- References tion of a sharp linkage peak is rare, and the identification of 1. MITOMAP. Human Mitochondrial Genome Database. Center for Molecular Med- linkage disequilibrium requires often screening of large num- icine Emory University, Atlanta, GA, USA, 2000; http://www.gen.emory.edu/mito- bers of markers in large numbers of patients and controls. We map.html. 2. Prezant TR, Agapian JV, Bohlman MC, Bu X, O¨ ztas S, Qiu W-Q, Arnos KS, were very fortunate that TDT testing revealed highly signifi- Cortopassi GA, Jaber L, Rotter JI, Shohat M, Fischel-Ghodsian N. 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Homoplasmic mitochondrial DNA diseases as the paradigm to share linkage disequilibrium with the same preferentially understand the tissue specificity and variable clinical severity of mitochondrial dis- orders. Mol Genet Metab 2000;71:93–99. transmitted allele is not surprising. First, it should be noted 6. Bykhovskaya Y, Shohat M, Ehrenman K, Johnson D, Hamon M, Cantor RM, Aouiz- that a NPL score Ͼ 0.8 does not necessarily mean that this erat B, Bu X, Rotter JI, Jaber L, Fischel-Ghodsian N. Evidence for complex nuclear family is linked to the locus. Second, even if all the families with inheritance in a pedigree with nonsyndromic deafness due to a homoplasmic mito- Ͼ chondrial mutation. Am J Med Genet 1998;77:421–426. NPL scores 0.8 were linked to the chromosome 8 locus, we 7. Bykhovskaya Y, Estivill X, Taylor K, Hang T, Hamon M, Casano RAMS, Yang H, may not yet have tested the right alleles to see full linkage dis- Rotter JI, Shohat M, Fischel-Ghodsian N. 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